Isolation and structure elucidation of cytotoxic secondary metabolites from valeriana species and investigation of their mechanisms of action

The genus Valeriana L. is rich in diverse group of bioactive secondary metabolites. The aim of this thesis was to purify potential cytotoxic compounds from the underground parts of V. alliariifolia Adams, V. sisymbriifolia Vahl. and V. speluncaria Boiss. through cytotoxicity-guided fractionation, and unravel their cell death mechanisms. The in vitro cytotoxic activity of crude ethanolic extracts prepared from Valeriana species were evaluated against human cancer cell lines including lung (A549), breast (MCF7), gastric (HGC27), prostate (PC3) and glioblastoma (U87) as well as a healthy cell line (HUVEC) by MTS assay. The crude EtOH extracts of V. alliariifolia and V. sisymbriifolia showed pronounced cytotoxic activity against A549, MCF7, HGC27 and PC3 cancer cells, whereas EtOH extract of V. speluncaria inhibited cell proliferation in U87 and MCF7 cells. The crude extracts were further partitioned to yield CHCl3, EtOAc, n-BuOH and rH2O subextracts. Chromatographic separations on the cytotoxic subextracts (CHCl3 and EtOAc) yielded 32 compounds, including six new ones, sisymbriifolivaltrate (17), sisymbriifolioside (18), sisymbriifolins A (22) and B (23), speluncarins A (30) and B (31). Their structures were elucidated by extensive NMR and HR-MS analyses. All the isolates were tested on the same cell lines and compounds 1-7 and 15-17 displayed remarkable cytotoxicity with IC50 values close to or less than 10 µM. The mechanisms behind the cytotoxic activities of the most potent ones, IVHD-valtrate (2), valtrate (3), 1-β-acevaltrate (4), seneciovaltrate (5), valtrate hydrine B2 (6), 1-α-acevaltrate (15), 7-homovaltrate (16) and sisymbriifolivaltrate (17) were evaluated with Annexin V/PI staining, DNA content analysis, and protein levels analysis by using flow cytometry. Compounds 2-6 and 15-17 induced cell death through different mechanisms including apoptosis, necrosis, autophagy and cell cycle arrest, depending on the cancer cell lines.